Selection and Validation of Novel RT-qPCR Reference Genes under Hormonal Stimuli and in Different Tissues of Santalum album

Haifeng Yan,Meiyun Niu,Yuan Li,Hanzhi Liang,Beiyi Guo,Yueya Zhang,Jaime A Teixeira Da Silva,Qingwei Chen,Xinhua Zhang,Guohua Ma,Mingzhi Li,Yuping Xiong

doi:10.1038/s41598-018-35883-6

Abstract

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a widely used technique to investigate gene expression levels due to its high throughput, specificity, and sensitivity. An appropriate reference gene is essential for RT-qPCR analysis to obtain accurate and reliable results. To date, no reliable reference gene has been validated for the economically tropical tree, sandalwood (Santalum album L.). In this study, 13 candidate reference genes, including 12 novel putative reference genes selected from a large set of S. album transcriptome data, as well as the currently used β-actin gene (ACT), were validated in different tissues (stem, leaf, root and callus), as well as callus tissue under salicylic acid (SA), jasmonic acid methyl ester (MeJA), and gibberellin (GA) treatments using geNorm, NormFinder, BestKeeper, Delta Ct and comprehensive RefFinder algorithms. Several novel candidate reference genes were much more stable than the currently used traditional gene ACT. ODD paired with Fbp1 for SA treatment, CSA and Fbp3 for MeJA treatment, PP2C and Fbp2 for GA treatment, as well as Fbp1 combined with Fbp2 for the total of three hormone treatments were the most accurate reference genes, respectively. FAB1A, when combined with PP2C, was identified as the most suitable reference gene combination for the four tissues tested, while the combination of HLMt, PPR and FAB1A were the most optimal reference genes for all of the experimental samples. In addition, to verify our results, the relative expression level of the SaSSy gene was evaluated by the validated reference genes and their combinations in the three S. album tissues and under MeJA treatment. The evaluated reference genes in this study will improve the accuracy of RT-qPCR analysis and will benefit S. album functional genomics studies in different tissues and under hormone stimuli in the future.

Highlights

Gene name FAB1A unknown function (UK) Fbp[1] CCS1 pentatricopeptide repeat-containing protein (PPR) coatomer subunit alpha-1 (CSA) Fbp[3] oxoglutarate-dependent dioxygenase (ODD) phosphatase 2 C (PP2C) histone-lysine N-methyltransferase ATXR3 (HLMt) Fbp[2] S8 actin gene (ACT) SaSSy
Only the housekeeping gene ACT was used as a reference gene in S. album, and no systematic validation and evaluation of stable reference genes for RT-Quantitative real-time PCR (qPCR) data normalization exists for the commercial S. album tree
A total of 12 novel genes were selected as candidate reference genes based on the coefficient of variation (CV) value calculated from transcriptome data

Summary

Introduction

Gene name FAB1A UK Fbp[1] CCS1 PPR CSA Fbp[3] ODD PP2C HLMt Fbp[2] S8 ACT SaSSy. GeneBank accession number MG282422 MG282423 MG282425 MG282427 MG282429 MG282432 MG282433 MG282424 MG282426 MG282428 MG282430 MG282431 EF452617 JX826486.1. As far as we known, the traditional housekeeping gene ACT (β-actin) was the only reference gene used to date[10,12,14,15], and there has been no systematic validation and evaluation of reference genes for RT-qPCR analysis in S. album. 13 candidate reference genes, including 12 novel genes selected from a large set of RNA-seq data in three different tissues (stem, leaf, and root) of S. album, as well as the currently used traditional housekeeping gene ACT, were assessed by RT-qPCR. This work validated a set of more stable novel reference genes and will facilitate, expand and fortify gene expression analysis in different tissues and under hormone treatment of S. album

Methods

Results

Conclusion